| Background&ObjectiveHepatocellular Carcinoma is one of the most frequent malignant tumors in adults,accounting for approximately650,000new cases per year in the world.Now, HCC is the third most common cause of cancer death.Although the biological characteristics and mechanisms of occurance and development of HCC became better understood,diagnostic techniques progressed markedly and surgical outcome for HCC has improved, the prognosis for HCC patients remains discouraging, with the recurrence of HCC as the main problem postoperatively.After radical resection of HCC, the5-year recurrence rate is more than65%.Thus,exploring the molecular mechanisms on tendency of recurrence and metastasis of HCC is of great importance for research on progression of HCC and prognosis estimation.Recently,studies found that C-IAP2(cellular inhibitor of apoptotic protein-2),as an important member of IAPs (inhibitor of apoptosis proteins), expressed highly in many kinds of tumor cells, which was highly associated with the occurance,development, recurrence and metastasis of tumors.In this study, the expressions of C-IAP2mRNA and its protein in three HCC cell lines with distinct aggressive phenotype were tested by quantitative real-time PCR western blot and immunofluorescence respectively; The expressions of C-IAP2mRNA and its protein in liver tumor,adjacent tumor tissues and non-cancer tissues were also detected by quantitative PCR and immunohistochemical staining, and the relationships between C-IAP2mRNA or C-IAP2protein and clinical pathological features were studied. Furthermore, the relationships between C-IAP2and recurrence and metastasis of HCC in tissues were analyzed to provide possible value for HCC in clinical diagnosis,progression and assessment of prognosis.Methods1、Quantitative real-time PCR, western blot and immunofluorescence were used to detect the expression C-IAP2mRNA and its protein in three HCC cell lines with distinct aggressive phenotype,including HepG2、SMMC7721and HCCLM3.2、All tissues were collected from the Department of Hepatobiliary Surgery ofNanfang Hospital in2010.11～2012.1.All samples were captured in vitro within2hours and were confirmed by pathology.3、Quantitative real-time PCR was used to detect the expression of C-IAP2mRNA in72cases with hepatocellular carcinoma, including tumor and non-cancer tissues, the relationships between C-IAP2mRNA and clinical pathological parameters were studied.4、The expression of C-IAP2protein was detected by immunohistochemical staining in75patients with hepatocellular carcinoma, including tumor, tumor surrounding and non-cancer tissues, the relationships between the expression of C-IAP2protein in tumor tissues and clinicopathological features were analysed.5、Statistical analysis:SPSS13.0software was used to analyse all data. The expression of C-IAP2protein in tumor, tumor surrounding and non-cancer tissues,and the relationships between C-IAP2protein and clinicopathological features were tested by Pearson Chi-square test. Comparisions of recurrence rate among different expression intension of C-IAP2mRNA or its protein in HCC were tested by survival analysis (Kaplan-Meier,log-rank).The data of Quantitative real-time PCR were handled with2-△△CT,each sample repeated trial for3times.then the mean of Ct and△CT value was calculated,measurement data was expressed by mean±SD.The expression of C-IAP2mRNA in tumor and non-cancer tissues, and the relationships between C-IAP2mRNA and clinical pathological parameters were compared by T-test,analysis of variance and LSD-t test.The expressions of C-IAP2mRNA and C-IAP2protein in three HCC cell lines with distinct aggressive phenotype were compared by One-Way ANOVA and LSD-t test. When P<0.05,the difference was considered statistically significant.Results1、The expression of C-IAP2mRNA in HCCLM3was54.95-fold higher than that in HepG2,and the expression in SMMC7721was12.13-fold higher than that in HepG2, the difference was statistically significant(P<0.05).2、The over expression of C-IAP2protein presented in HCCLM3(9.109±0.632), the moderate expression of C-IAP2protein was in SMMC7721(2.689±0.637), and the low expression of C-IAP2protein did in HepG2(0.297±0.556), the difference was statistically significant (F=348.001; P<0.001)3、C-IAP2protein detected by Immunofluorescence was found in cytoplasm and nucleus of the three HCC cell lines. The over expression of C-IAP2protein expressed in HCCLM3, the moderate expression of C-IAP2protein presented in SMMC7721, and the low expression of C-IAP2protein was in HepG2.4、The expression of C-IAP2mRNA in tumor tissues was2.70-fold higher than that in non-cancer tissues, the difference was statistically significant(P<0.001).5、The expression of C-IAP2mRNA in HCC was associated with tumor thrombosis, AFP level,lymph node metastasis, histological differentiation type, TNM stage and prognosis (P<0.05), but not with patients’gender, age, HbsAg positivity, tumor size, number of tumors, cirrhosis and the presence of tumor encapsulation (P>0.05).The expression of C-IAP2mRNA in patients with tumor thrombosis and lymph node metastasis was9.65-fold and6.53-fold higher than those without tumor thrombosis and lymph node metastasis respectively, patients with AFP>400ug/L was2.79-fold higher than those with AFP<400ug/L.In TNM stage, the expression in Ⅲ-Ⅳ stage was6.63-fold higher than that in Ⅰ-Ⅱ stage.Expressions in moderate differentiation and low differentiation were not statistically different,but both were higher than that in high differentiation. The recurrence rate in group with high expression of C-IAP2mRNA was higher than that in low expression group.6、C-IAP2protein expressed in intracytoplasm,the positive rate of C-IAP2protein in tumor surrounding tissues, cancer tissues and non-cancer tissues was86.7%,69.3%and26.7%, respectively.(χ2=60.057,P<0.001)7、The expression level of C-IAP2in hepatocellular carcinoma was related to the tumor differentiation, TNM stage, AFP value, lymph node metastasis,tumor thrombi and prognosis (P<0.05), the expression level of C-IAP2in tumor tissues increased with the reduction of the tumor differentiation and whose with AFP≥400ug/L were higher than those with AFP<400ug/L,the expression level of C-IAP2in patients with tumor thrombi and lymph node metastasis was higher than those without tumor thrombi and lymph node metastasis, the expression in stage Ⅲ-Ⅳ was higher than that in stage Ⅰ-Ⅱ, The recurrence rate in group with high expression of C-IAP2protein was higher than that in low expression group,which was higher than that in negative group.Conclusions 1、C-IAP2mRNA and its protein expressed in the three HCC cell lines with distinct aggressive phenotype, the over expression of C-IAP2mRNA and its protein presented in HCCLM3, the moderate expression was in SMMC7721, and the low expression did in HepG2.The expression levels of C-IAP2mRNA and its protein were related to the invasion of HCC cell lines.2、The expression of C-IAP2mRNA in tumor tissues was significantly higher than that in non-cancer tissues. C-IAP2protein expressed in intracytoplasm, the positive rate of C-IAP2protein in tumor surrounding tissues, cancer tissues and non-cancer tissues was86.7%,69.3%and26.7%, respectively.The expression of C-IAP2mRNA and its protein in HCC was associated with tumor thrombosis, lymph node metastasis, histological differentiation type, AFP level, TNM stage and prognosis, but not with patients’other clinical features.These indicated that C-IAP2may influence biological characteristics of HCC.3、The expression levels of C-IAP2mRNA and its protein increased as the aggression of HCC cell lines raised; Also.The expressions of C-IAP2mRNA and its protein in liver tumors were higher than that in non-cancer tissues,and the expression level in HCC was closely associated with histological differentiation type and prognosis. C-IAP2may be related to progression and prognosis of HCC,it may become the prognosis biological marker or potential new therapeutic target for HCC. |
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